PROJECT SUMMARY Natural or thymic regulatory T cells (tTregs) are a subpopulation of CD4+ lymphocytes that develop in the thymus to maintain self-tolerance, thereby preventing the development of immune-mediated disease and autoimmune disorders. Moreover, aberrant activity of Tregs can suppress antitumor immune activity and exacerbate the growth and/or metastasis of certain tumors. Consequently, understanding the mechanisms that drive tTreg development and function will inform new approaches for controlling autoimmune diseases and immunity to certain cancers. Most autoreactive T cells that develop in the thymus die by apoptosis following contact with high affinity antigen/agonists by a process termed negative selection. However, a small proportion of these cells is diverted from apoptotic death to become tTregs. Development of tTregs is critically regulated by the expression and then activation of the transcription factor c-Rel. Indeed, a block in tTreg development in c-Rel-deficient mice is associated with defective generation of tTreg precursors. However, our inability to visualize c-Rel expression in live cells is a major roadblock to defining how this is controlled by antigen and performing lineage tracing experiments on c-Rel+ tTreg precursors. Therefore, in this high impact R21 application, we outline strategies to develop c-Rel reporter mice to facilitate studies of the relationship between TCR signaling, c-Rel expression, and genetic mechanisms by which c-Rel drives commitment of autoreactive T cell clones to tTreg development. While control of c-Rel expression represents an early checkpoint in nTreg development, very little is known about the subsequent mechanisms that regulate c-Rel activation and transcriptional specificity. Our exciting new data provide an original conceptual framework to explore this question. Our unbiased mass spectrometry (MS) experiments revealed Serine 34 as a novel target on c-Rel that undergoes TCR-induced, Ca2+-dependent phosphorylation. Furthermore, preliminary analysis of a new mouse line with an alanine substitution at c-Rel S34 reveals a crucial role for phosphorylation of this residue in tTreg development. Indeed, preliminary RNAseq analysis indicate that c-Rel S34 phosphorylation tunes the pattern of gene expression in T cells. In this proposal, we outline an approach using these mice to explore general mechanisms of tTreg development and specifically to dissect how phosphorylation of c-Rel at S34 regulates commitment to nTreg development. The conceptually and technically innovative experiments we propose using the mice we have and will generate will yield important therapeutic insights and reveal new targets for regulating the susceptibility to and course of immune-mediated diseases.